Studies of insect peptides alloferon, Any-GS and their analogues. Synthesis and antiherpes activity

Article abstract of DOI:10.1002/psc.1219

The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as alloferon, Any-GS and their analogues. The synthesis of 14 peptideswas performed by the solid-phase method. Biological effect of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV-1) in vitro using a Vero cell line. It was found that the investigated peptides inhibit the replication of HHV-1 in Vero cells. Copyright

Full text of DOI:10.1002/psc.1219

Research Article
Received: 15 October 2009
Revised: 29 December 2009
Accepted: 21 January 2010
Published online in Wiley Interscience: 26 February 2010
(
www.interscience.com) DOI 10.1002/psc.1219
Studies of insect peptides alloferon, Any-GS
and their analogues. Synthesis and antiherpes
activity
a∗
a
b
Mariola Kuczer, Katarzyna Dziubasik, Anna Midak-Siewirska,
b
b
a
Renata Zahorska, Mirosław Łuczak and Danuta Konopi n´ ska
The subject of these studies was synthesis and determination of biological properties of a series of insect peptides, such as
alloferon, Any-GS and their analogues. The synthesis of 14 peptides was performed by the solid-phase method. Biological effect
of these peptides was evaluated by the antiviral test against Human Herpes Virus type 1 (HHV-1) in vitro using a Vero cell line.
It was found that the investigated peptides inhibit the replication of HHV-1 in Vero cells. Copyright ꢀc 2010 European Peptide
Society and John Wiley & Sons, Ltd.
Keywords: alloferon; insect peptides; antiviral peptides; antiviral activity; Human Herpes Virus
Introduction
cause skin and mucous membranes infections, tumor, and other
serious diseases in animals and humans. A major problem of the
modern medicine is a relatively small number of efficient antiviral
drugs. Therefore, the identification of new biologically active
substanceswhichmaybeusedasantiviralagentsisveryimportant
The aim of the present study was to search for new antiviral activity
among selected insect peptides such as alloferon, Any-GS and a
series of their analogs.
Alloferon is a tridecapeptide (H-His-Gly-Val-Ser-Gly-His-Gly-
Gln-His-Gly-Val-His-Gly-OH) isolated from the bacteria-challenged
larvae of the blow fly Calliphora vicina [1]. It is interesting that
its primary structure is similar to some functionally relevant
proteins, such as influenza virus B hemagglutinin, bovine prion
protein I and II, and Sarcophaga peregrina antifungal protein.
The in vitro experiments reveal that alloferon has a stimulatory
activity toward natural killer lymphocytes [1]. In vivo, alloferon
induces the IFN synthesis in mice [1]. A further study has shown
that alloferon stimulates the synthesis of IFN through NF-κB
activation [2]. Additional in vivo experiments in mice indicate that
alloferon has both antiviral and antitumor capabilities [1]. Due to a
unique immune modulating mechanism, alloferon may be used in
conjunction with other antiviral and anticancer drugs to generate
synergistic effects in combating disease.
[
8]. Natural products isolated from arthropods are a significant
sourceofbioactivecompounds.Inthelastdecade,severalpeptides
with a large variety of biological activities were isolated from
insects[1,4,9–12]. Thesepeptidesshow, amongothers, myotropic,
oostatic, antinociceptive, antimicrobial, and antitumor activity
[9–12]. However, relatively little data are available on molecules
from insects with the antiviral properties. Recently, several new
peptides with antiviral or antitumor activity, such as: alloferon,
Any-GS, N-myristoylated peptide and red fluorescent protein have
been isolated from insects [11], opening new possibilities in the
development of the much requested antiviral substances. Because
both alloferon and Any-GS show a broad-spectrum activity [1,4],
it inspired us to investigate their influence on the growth of the
herpesviruses. Moreover, the insight into the relation between
The oligopeptide Any-GS (H-Asp-Ile-Leu-Arg-Gly-NH2) was
isolated from the wild silkmoth Antheraea yamamai [3]. This
peptidesuppressestheproliferationofrathepatomacells(dRLh84)
in vitro [4]. Furthermore, it provokes the inhibition of growth and
viability of human hepatoma cells HepG2 and decreases the
activity of mitochondrial enzymes [5]. The mode of antitumor
action of this molecule is still unknown; however, the biological
study has shown that Any-GS causes the cell cycle arrest rather
than apoptosis/necrosis [4]. Such evidence puts this peptide
as a potentially interesting chemipharmaceutical. In addition,
Any-GS shows several other biological activities. For example,
Any-GS inhibits in vitro the growth of plant pathogens Phoma
narcissi and Botrytis tulipae [6]. Moreover, Any-GS shows a strong
cardioinhibitory effect in the myotropic test on the semi-isolated
heart of insect Tenebrio molitor [7].
∗
Correspondence to: Mariola Kuczer, Faculty of Chemistry, University of
Wroclaw, 14 F. Joliot-Curie Street, 50-383 Wroclaw, Poland.
E-mail: km@wchuwr.chem.uni.wroc.pl
a Faculty of Chemistry, University of Wroclaw, 14 F. Joliot-Curie Street, 50-383
Wroclaw, Poland
b Department of Microbiology, Medical University of Warsaw, 5 Chałubi n´ skiego
Street, 02-004 Warsaw, Poland
Abbreviations used: CPE, cytopathic effect; DMF, dimethylformamide; DNA,
deoxyribonucleic acid; FBS, fetal bovine serum; HBTU, O-benzotriazole-
ꢁ
ꢁ
N,N,N ,N -tetramethyl-uronium-hexafluorophosphate; HHV, Human Her-
pes Virus; HHV-1MC, Human Herpes Virus type 1 McIntrie; HOBt, N-
hydroxybenxotriazole; HPLC, high performance liquid chromatography; INF,
interferon; NEM, N-ethylmorpholine; NF, nuclear factor; MTT, 3-[4,5-dimethyl-
thiazol-2-yl]-2,5-diphenyl tetrazolium bromide; TCID, tissue culture infected
dose; TFA, trifluoroacetic acid; TLC, thin layer chromatography; UV, ultraviolet.
One of the most common viral infections in humans is caused by
HHV. The Herpesviridae are a large family of DNA viruses that can
J. Pept. Sci. 2010; 16: 186–189
Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd.

ANTIHERPES ACTIVITIES OF SELECTED INSECT PEPTIDES
Table 1. Physicochemical data of alloferon and its analogues modified at position 1
Rf (TLC)b
[
α]20
c = 1, methanol (HPLC)
D
Rt
Molecular weight Molecular weight
Peptide
Yield (%)a
78
calculated
found
X
Y
Z
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-
Gly-Val-His-Gly-OH (1)
−28.5
−18.3
−21.4
−17.8
−14.4
11.2
12.2
12.8
11.7
11.3
1264.5
1265.5
0.32 0.32 0.25
0.33 0.34 0.26
0.33 0.35 0.28
0.30 0.33 0.26
0.36 0.31 0.22
H-Lys-Gly-Val-Ser-Gly-His-Gly-Gln-His-
Gly-Val-His-Gly-OH (2)
76
81
85
80
1255.7
1283.7
1198.6
1127.5
1256.6
1284.5
1199.6
1128.5
H-Arg-Gly-Val-Ser-Gly-His-Gly-Gln-His-
Gly-Val-His-Gly-OH (3)
H-Ala-Gly-Val-Ser-Gly-His-Gly-Gln-His-
Gly-Val-His-Gly-OH (4)
H-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-
Val-His-Gly-OH (5)
a
Crude yield: yield after cleavage of the resin. The purity of crude product was analyzed according to HPLC peak integrals at λ 210 nm on analytical
HPLC. The crude peptide had a purity of >80%.
TLC on silica gel plates, eluents : X = n-butanol : acetic acid : methanol (4 : 1 : 1), Y = chloroform : methanol : acetic acid (5 : 3 : 1), Z = n-butanol :
b
pyridine : acetic acid : water (30 : 20 : 6 : 24).
the peptide structure and function was applied to investigate the Material and Methods
mechanism of action of selected insect antiviral peptides as well
Peptides Synthesis
as in the design of their analogues as potential novel therapeutic
agents.
Taking into account the frequent occurrence of histidines and
Peptides were prepared by the manual solid-phase techniques.
The synthesis of peptides 1–5 was performed using the
standard Fmoc procedure on a Wang resin. Other peptides
(6–14) were synthesized by the classical solid-phase method
according to the Boc-procedure [7]. The Wang resin preloaded
with Fmoc-Gly (capacity 0.84 mmol/g) was purchased from
Novabiochem. The 9-fluorenyl-methoxy-carbonyl amino acids,
the coupling reagent HBTU and HOBt were purchased from
Novabiochem. The side chain protecting groups for Fmoc-
amino acids were t-butyl for Ser, trityl for His and Gln, and
t-butoxycarbonyl for Lys. NEM was purchased from Fluka. TFA
and N,N-dimethylformamide were obtained from Iris Biotech.
HPLC-grade solvents were purchased from Fisher Scientific. All
other reagents were purchased from Sigma-Aldrich (Steinheim,
Germany). All solvents and reagents used for solid-phase synthesis
were of analytical quality and were used without further
purification.
aspartates in the active or binding sites of proteins [13–15], in our
preliminary studies we decided to obtain information about the
1
1
role of the His of alloferon and the Asp of Any-GS.
We performed the synthesis of the following peptides:
1
2
.
.
alloferon (H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-
Gly-OH) (1) and its analogues modified at position 1: H-Lys-
Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH (2), H-Arg-
Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH (3), H-Ala-
Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH (4), H-Gly-
Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH ( 5),
Any-GS (H-Asp-Ile-Leu-Arg-Gly-NH2) (6) and its analogues
modified at position 1, such as: H-Arg-Ile-Leu-Arg-Gly-NH2
(
(
7), H-Gly-Ile-Leu-Arg-Gly-NH2 (8), H-Ala-Ile-Leu-Arg-Gly-NH2
9), H-Asn-Ile-Leu-Arg-Gly-NH2 (10), H-Gln-Ile-Leu-Arg-Gly-
Peptides were purified by the preparative HPLC using a Varian
ProStar system with a column: Tosoh Bioscience ODS-120T C18
NH2 (11) and its shortened derivatives: H-Asp-Ile-Leu-Arg-NH2
12), H-Ile-Leu-Arg-Gly-NH2 (13), H-Leu-Arg-Gly-NH2 (14).
(
(
ODS300×21.5 mm)inalineargradientof15–30%BinA(A – 0.1%
aqueous TFA, B – 80% acetonitrile in water, containing 0.1% TFA)
for 40 min at a flow rate of 7 ml/min with the UV detection at
In the first group of peptides, the His residue at position 1 of
alloferon was replaced by: (i) basic aliphatic amino acid – Lys (2)
or Arg (3), (ii) hydrophobic aliphatic amino acid – Ala (4). We also
obtained the analog without the His residue at position 1 ( 5).
In the next group of investigated peptides the Asp residue at
position 1 of Any-GS was exchanged by: basic amino acid – Arg
2
10 nm.
Analytical HPLC was performed on a Thermo Separation
Products HPLC system with a Vydac C18 column (ODS 250 ×
4
.6 mm) with a linear gradient 0–100% B in A for 60 min at a flow
rate of 1 ml/min with the UV absorption determined at 210 nm.
The molecular weight of the peptides was determined with a
Bruker Daltonics micrOTOF-Q mass spectrometer.
The optical activity of the chiral compounds was measured with
a Jasco DIP-1000 polarimeter (Jasco).
(7), neutral amino acid – Gly (8) or Ala (9) and neutral hydrophilic
amino acid – Asn (10) or Gln (11). Moreover, we synthesized
truncated analogues of Any-GS in which one or two amino acids
were eliminated at the C-(12) or the N-terminus (13–14).
Peptides 1–5 were synthesized by the classical solid-phase
method according to the Fmoc procedure. Their physicochemical
data are presented in Table 1. Other peptides (6–14) were
synthesized by use of the Boc solid-phase chemistry, according to
the previously described procedure [7].
TLC was performed on aluminum sheets precoated with silica
gel 60 from Merck.
H-His-Gly-Val-Ser-Gly-His-Gly-Gln-His-Gly-Val-His-Gly-OH (1)
During the biological investigations of those peptides, we
examined their in vitro antiviral activity against HHV-1MC and
cytotoxic activity using a Vero cell line.
The peptide was obtained by a stepwise elongation of the peptide
chain by the method outlined above. 0.5 g of the Fmoc-Gly-
resin (capacity 0.84 mmol/g) was suspended in 20% solution of
J. Pept. Sci. 2010; 16: 186–189 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc

KUCZER ET AL.
piperidine in DMF. The mixture was stirred for 20 min at room
temperature. Then it was filtered and washed with DMF. The next
amino acid, Fmoc-His(Trt)-OH (0.826 g, 3 equiv), was dissolved in
DMF and coupled to the resin in the presence of HBTU (1.352 g,
Belgium) [16]. The stock solutions of peptides were prepared in
sterile water and stored at 4 C. Their concentrations are presented
◦
in Tables 2 and 3. Vero cells were inoculated in 96-microwell
plate. After incubation for 24 h, the peptides in the serial twofold
dilutions from 1 : 4 to 1 : 128 were added to the culture medium,
and cultured for further 24 or 48 h. The control was prepared
without any sample. All experiments were performed in triplicate.
The toxicity was calculated as a per cent of the control.
3
equiv), HOBt (0.180 g, 3 equiv) and NEM (293 µl, 6 equiv)
for 2 h. The end of the reaction was determined by the Kaiser
test. Other Fmoc-amino acid derivatives: Fmoc-Val-OH (0.453 g, 3
equiv), Fmoc-Gly-OH (0.397, 3 equiv), Fmoc-His(Trt)-OH (0.826 g, 3
equiv), Fmoc-Gln(Trt)-OH (0.815 g, 3 equiv), Fmoc-Gly-OH (0.397, 3
equiv), Fmoc-His(Trt)-OH (0.826 g, 3 equiv), Fmoc-Gly-OH (0.397, 3
Antiviral assay
t
equiv), Fmoc-Ser(Bu )-OH(0.512 g, 3equiv), Fmoc-Val-OH(0.453 g,
3
equiv), Fmoc-Gly-OH (0.397, 3 equiv) and Fmoc-His(Trt)-OH
The antiviral activity was assessed against HHV-1MC in vitro using a
(
0.826 g, 3 equiv) were connected to the resin in the same way.
Vero cell line. In these studies the cell cultures were infected with
α
After the final removal of the N -Fmoc group, the peptide-resin
was washed with DMF, MeOH : DMF (1 : 1, v/v), MeOH and then
dried overnight over KOH under reduced pressure. The free
peptide was obtained by deprotection with 4.75 ml of TFA in
the presence of 0.125 ml of ethanedithiol and 0.125 ml of water at
room temperature according to the standard procedure. Then the
peptide was purified by the preparative HPLC. The main fractions
were combined and lyophilized. The purity and homogeneity of
all final products were checked by HPLC, TLC, optical activity and
molecular weight determinations. Peptides 2–5 were obtained
and purified in the same manner as peptide 1. Their analytical data
are presented in Table 1.
HHV-1MC 1TCID /cell. The tested compounds were added at their
50
−
5
maximum non-toxic concentrations (∼10 M) to the cell culture
withthevirus. Theantiviralactivityofthepeptideswasdetermined
initially using a CPE. The inhibition of the viral CPE was assessed
by a light microscopy. Virus titers were determined according to
the Reed–Muench formula [16] and expressed in TCID / ml at the
5
0
particular stages of the experiments. The antiviral activity of the
investigated peptides was finally expressed as a reduction ratio of
the virus titers by comparison with the virus control.
Results and Discussion
The antiviral bioassay shows that all the investigated peptides
evidently inhibit the replication of HHV-1MC virus in vitro at their
maximum non-toxic concentrations after 24 and 48 h (Tables 2
and 3).
We found that alloferon at the concentration of 90 µg/ml
inhibits in vitro the replication of virus HHV-1MC by 2 log after
Biological Investigation
Cell culture and viruses
Vero cells were grown and maintained in the Eagle’s minimum
essential medium 1959 (Biomed Lublin) supplemented with 10%
FBS from Gibco and 1% of antibiotic antimycotic solution (100×):
◦
24 h of incubation but its inhibitory effect after 48 h of incubation
penicilin, streptomycin, amfotericin B (Sigma-Aldrich) at 37 C.
is weaker (1.5 log). A similar activity was observed after 24 h
for analogs of alloferon modified at position 1 (peptides 2–5).
However, these peptides after 48 h of incubation demonstrate a
weak inhibitory effect on the replication of virus HHV-1.
Interesting results in the antiviral test were also observed in the
case of the second group of investigated peptides.
The viral strain used in this study was HHV-1MC.
The virus stock was grown on Vero cells. After a CPE was
evident, the cells were frozen-thawed several times. The cell debris
was removed by centrifugation. The supernatant was aliquoted,
◦
titrated, and kept at −70 C. In the antiviral assay, the medium was
supplemented with 2% FBS and the above mentioned antibiotics.
Any-GS(6)attheconcentrationof52.5 µg/mlinhibitsin vitrothe
replication of HHV-1MC after 24 and 48 h by 1.5 and 1 log, respec-
tively (Table 3). The analogues of Any-GS modified at position 1 of
the peptide chain (peptides 7–11), in which the N-terminal Asp
residuewasexchangedbyArg, Gly, Ala, AsnorGln, reducethevirus
titer by 2 to 1 log after 24 h. After 48 h of incubation, this effect is
becoming weaker and the peptides reduce the virus titer by 0.5 to
Cytotoxicity
The cytotoxic activity of peptides was assessed by a light
microscopy (Olympus CK2) and quantified by the MTT (ATCC
bioproducts) assay in vitro using a Vero cell line. The absorbance
was read at 405 nm (Reader 230, Organon Teknika Turnhout,
Table 2. Influence of alloferon and its analogues on the replication of the HHV-1MC virus in vitro
Effect after 24-h incubation
Effect after 48-h incubation
Concentration
µg/ml)
Virus titer
(TCID50/ ml)
Virus titer
(TCID50/ ml)
(
Inhibition
(logTCID50/ ml)
Inhibition
(logTCID50/ ml)
Peptide
Stock solution
MNCa
Control
Sample
Control
Sample
3
3
2
4
2
1
2
3
4
5
360
155
160
150
150
90
8 × 10
1.1 × 10
7 × 10
2
1.8 × 10
8 × 10
1.5
1
3
3
3
3
2
38.7
40
1.7 × 10
1.7 × 10
1.7 × 10
1.7 × 10
1.5
2
3 × 10
3 × 10
3 × 10
3 × 10
3 × 10
3
3
3
2
1.3 × 10
3 × 10
3 × 10
1
2
37.5
37.5
2
3 × 10
1
2
0.7 × 10
2.5
3 × 10
1
a
Maximal non-toxic concentration.
www.interscience.com/journal/psc Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. J. Pept. Sci. 2010; 16: 186–189

ANTIHERPES ACTIVITIES OF SELECTED INSECT PEPTIDES
Table 3. Influence of Any-GS and its analogues on the replication of the HHV-1MC virus in vitro
Effect after 24-h incubation
Effect after 48-h incubation
Concentration
µg/ml)
Virus titer
(TCID50/ ml)
Virus titer
(TCID50/ ml)
(
Inhibition
(logTCID50/ ml)
Inhibition
(logTCID50/ ml)
Peptide
Stock solution
MNCa
Control
Sample
Control
Sample
3
3
2
4
3
6
7
8
9
210
250
135
160
195
105
230
206
230
52.5
62.5
33.7
40
8 × 10
3 × 10
1.5
2
1.8 × 10
1 × 10
2.1 × 10
1.1 × 10
1
3
3
3
3
3
4
2
2
1.7 × 10
1.7 × 10
1.7 × 10
1.7 × 10
1.7 × 10
1.6 × 10
3 × 10
3 × 10
3 × 10
3 × 10
3 × 10
3 × 10
1
3
3
3
3
2
1
2
2
2.8 × 10
7 × 10
2
8 × 10
1.5
0.5
1
1
1
1
1
1
0
1
2
3
4
48.8
26.3
57.5
51.5
57.5
1.5
2
8.3 × 10
2
3 × 10
3 × 10
3 × 10
5 × 10
3
3
3
2
2
3
2
2
3
8 × 10
8 × 10
8 × 10
1.8 × 10
1.3 × 10
1.2 × 10
1.5
2
1.8 × 10
1.8 × 10
1.8 × 10
2
4
4
1.5
1.5
1
1.1 × 10
a
Maximal non-toxic concentration.
1
log. It is interesting that the truncated analogues of Any-GS with-
2 Ryu MJ, Anikin V, Hong SH, Jeon H, Yu YG, Yu MH, Chernysh S, Lee C.
Activation of NF-kappaB by alloferon through down-regulation of
antioxidant proteins and IkappaBalpha. Mol. Cell. Biochem. 2008;
out the C-terminal Gly (12), N-terminal Asp (13) or the fragment
Asp-Ile (14) preserved the antiviral properties of native Any-GS.
These results can indicate that the C-terminal amino acid
residuesofAny-GSareimportantfortheantiviralactivity. However,
the deletion of the first two residues of Any-GS resulted in a loss
of the cell growth suppression activity on dRLh84 cells [4]. A more
detailed study to clarify this effect is in progress.
Moreover, alloferon, Any-GS, and their investigated analogs did
not show any cytotoxic activity against the Vero cells at tested
concentrations.Microscopicobservationsshowedthatnochanges
occurred in the Vero cell growth or morphology in the presence
of the tested peptides. The MTT assay also proved that they had
no effect on the cell proliferation.
3
13: 91–102.
3
Suzuki K, Minagawa T, Kumagai T, Naya S, Endo Y, Osanai M,
Kuwano E. Control mechanism of diapause of the pharate first-
instar larvae of the silkmoth Antheraea yamamai. J. Insect Physiol.
1990; 36: 855–860.
4
Yang P, Abe S, Zhao YP, An Y, Suzuki K. Growth suppression of rat
hepatoma cells by a pentapeptide from Antheraeayamamai. J.Insect
Biotech. Sericol. 2004; 73: 7–13.
5 Słoci n´ ska M, Olejnik A, Kuczer M. Insect peptides-new perspective
for cancer therapy? Acta Biochim. Polon. 2008; 54: 135–135.
6
7
8
9
Kuczer M, Dziubasik K, Łuczak M, Majewska A, Kamysz W,
Saniewska A, Konopi n´ ska D. The search for new biological activities
for selected insect peptides. Pestycydy 2008; 1–2: 5–11.
Szymanowska-Dziubasik K, Marciniak P, Rosi n´ ski G, Konopi n´ ska D.
Synthesis, cardiostimulatory, and cardioinhibitory effects of selected
insect peptides on Tenebrio molitor. J. Pept. Sci. 2008; 14: 708–713.
Dziecia ¸ t kowski T, Rola A, Majewska A, Solarska M, Łuczak M. Drugs
used in antiherpesviral therapy in humans. Post. Mikrobiol. 2007; 46:
211–221.
Conclusions
Based on the biological results presented here we can conclude
that:
Bulet P, St o¨ cklin R. Insect antimicrobial peptides: structures,
properties and gene regulation. Protein Pept. Lett. 2005; 12: 3–11.
•
•
•
•
The investigated peptides strongly inhibit the replication of
HHV-1 in Vero cells.
The presence of hydrophilic residues at position 1 of alloferon
is not important for inhibition of the virus replication.
The basic or acidic character of residues at position 1 of Any-GS
does not affect the antiviral activity.
No cytotoxic activity was observed for all tested compounds
against the Vero cells.
Theseeffectsconstituteabasisforfuturestudiesontheantiviral
activity of insect peptides and may lead to development of
novel antiviral drug.
10 Kuczer M, Rosi n´ ski G, Konopi n´ ska D. Insect gonadotropic peptide
hormones: some recent developments. J. Pept. Sci. 2007; 13: 16–26.
1
1 Słoci n´ ska M, Marciniak P, Rosi n´ ski G. Insects antiviral and anticancer
peptides: new leads for the future? Protein Pept. Lett. 2008; 15:
578–585.
12 G a¨ de G,Goldsworthy JG.Insectpeptidehormones:aselectivereview
of their physiology and potential application for pest control. Pest.
Manag. Sci. 2003; 59: 1063–1075.
1
3 Docherty JJ, Pollock JJ. Inactivation of herpes simplex virus types 1
and 2 by synthetic histidine peptides. Antimicrob. Agents Chemother.
1987; 31: 1562–1566.
14 Pourmotabbed T, Aelion JA, Tyrrell D, Hasty KA, Bu CH, Mainardi CL.
Role of the conserved histidine and aspartic acid residues in activity
and stabilization of human gelatinase B: an example of matrix
metalloproteinases. J. Protein Chem. 1995; 14: 527–535.
Acknowledgements
1
5 Guan R, Roderick SL, Huang B, Cook PF. Roles of histidines 154 and
89 and aspartate 139 in the active site of serine acetyltransferase
1
This work was supported by the University of Wroclaw grant
2
AM20/WA and W2/2008–2009.
from Haemophilus influenzae. Biochemistry 2008; 47: 6322–6328.
6 Reed LJ, Muench HA. A simple method of estimating fifty per cent
endpoint. Am. J. Hyg. 1938; 27: 493–497.
237/W/WCh/09 and by the Medical University of Warsaw grants
1
References
1
Chernysh S, Kim SI, Bekker G, Pleskach VA, Filatova NA, Anikin VB,
Platonov VG, Bulet P. Antiviral and antitumor peptides from insects.
PNAS 2002; 99: 12 628–12 632.
J. Pept. Sci. 2010; 16: 186–189 Copyright ꢀc 2010 European Peptide Society and John Wiley & Sons, Ltd. www.interscience.com/journal/psc